Numerous diseases are thought to be initiated by disruptions in genomic stability. For example, sickle cell anemia, phenylketonuria, hemophilia, cystic fibrosis, and various cancers have been associated with one or more genetic mutation(s). Increased knowledge of the molecular basis for disease has lead to a proliferation of screening assays capable of detecting disease-associated nucleic acid mutations.
One such method identifies a genomic region thought to be associated with a disease and compares the wild-type sequence in that region with the sequence in a patient sample. Differences in the sequences constitute a positive screen. See e.g., Engelke, et al., Proc. Natl. Acad. Sci., 85: 544-548 (1988). Such methods are time-consuming, costly, and often results in an inability to identify the mutation of interest. Thus, sequencing is not practical for large-scale screening assays.
A variety of detection methods have been developed which exploit sequence variations in DNA using enzymatic and chemical cleavage techniques. A commonly-used screen for DNA polymorphisms consists of digesting DNA with restriction endonucleases and analyzing the resulting fragments by means of Southern blots, as reported by Botstein etal., Am. J. Hum. Genet., 32: 314-331 (1980) and White et al., Sci. Am., 258: 40-48 (1988). Mutations that affect the recognition sequence of the endonuclease will preclude enzymatic cleavage at that site, thereby altering the cleavage pattern of the DNA. Sequences are compared by looking for differences in restriction fragment lengths. A problem with this method (known as restriction fragment length polymorphism mapping or RFLP mapping) is its inability to detect mutations that do not affect cleavage with a restriction endonuclease. One study reported that only 0.7% of the mutational variants estimated to be present in a 40,000 base pair region of human DNA were detected using RFLP analysis. Jeffreys, Cell, 18: 1-18 (1979).
Single-base mutations have been detected by differential hybridization techniques using allele-specific oligonucleotide probes. Saiki et al., Proc. Nati. Acad. Sci., 86: 6230-6234 (1989). Mutations are identified on the basis of the higher thermal stability of the perfectly-matched probes as compared to mismatched probes. Disadvantages of this approach for mutation analysis include: (1) the requirement for optimization of hybridization for each probe, and (2) the nature of the mismatch and the local sequence impose limitations on the degree of discrimination of the probes. In practice, tests based only on parameters of nucleic acid hybridization function poorly when the sequence complexity of the test sample is high (e.g., in a heterogeneous biological sample). This is partly due to the small thermodynamic differences in hybrid stability generated by single nucleotide changes. Therefore, nucleic acid hybridization is generally combined with some other selection or enrichment procedure for analytical and diagnostic purposes.
A number of detection methods have been developed which are based on template-dependent, primer extension. Those methods can be placed into one of two categories: (1) methods using primers which span the region to be interrogated for the mutation, and (2) methods using primers which hybridize upstream of the region to be interrogated for the mutation.
In the first category, U.S. Pat. No. 5,578,458 reports a method in which single base mutations are detected by competitive oligonucleotide priming under hybridization conditions that favor the binding of a perfectly-matched primer as compared to one with a mismatch. U.S. Pat. No. 4,851,331 reports a similar method in which the 3' terminal nucleotide of the primer corresponds to the variant nucleotide of interest. Since mismatching of the primer and the template at the 3' terminal nucleotide of the primer inhibits elongation, significant differences in the amount of incorporation of a tracer nucleotide result under normal primer extension conditions.
Methods in the second category are based on incorporation of detectable, chain-terminating nucleotides in the extending primer. Such single nucleotide primer-guided extension assays have been used to detect aspartylglucosaminuria, hemophilia B, and cystic fibrosis; and for quantifying point mutations associated with Leber Hereditary Optic Neuropathy. See. e.g., Kuppuswamy et al., Proc. Natl. Acad. Sci. USA, 88: 1143-1147 (1991); Syvanen et al., Genomics, 8: 684-692 (1990); Juvonen et al., Human Genetics, 93: 16-20 (1994); Ikonen et al., PCR Meth. Applications, 1: 234-240 (1992); Ikonen et al., Proc. Natl. Acad. Sci. USA, 88: 11222-11226 (1991); Nikiforov et al., Nucleic Acids Research, 22: 4167-4175 (1994). An alternative primer extension method involving the addition of several nucleotides prior to the chain terminating nucleotide has also been proposed in order to enhance resolution of the extended primers based on their molecular weights. See e.g., Fahy et al., WO/96/30545 (1996).
Strategies based on primer extension require considerable optimization to ensure that only the perfectly annealed oligonucleotide functions as a primer for the extension reaction. The advantage conferred by the high fidelity of the polymerases can be compromised by the tolerance of nucleotide mismatches in the hybridization of the primer to the template. Any "false" priming will be difficult to distinguish from a true positive signal. The reaction conditions of a primer extension reaction can be optimized to reduce "false" priming due to a mismatched oligonucleotide. However, optimization is labor intensive and expensive, and often results in lower sensitivity due to a reduced yield of extended primer.
A number of mutations leading to various forms of cancer involve the deletion of multiple nucleotides from a genomic sequence. An example is the BAT26 segment of the MSH2 mismatch repair gene. The BAT26 segment contains a long poly-A tract. In certain cancers, a characteristic 5 base pair deletion occurs in the poly-A tract. Detection of that deletion may provide diagnostic information. Accordingly, the invention provides methods for detecting deletions in genomic regions, such as BAT26 and others, which may be associated with disease.